Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Mechanisms by Which Dietary Fatty Acids Regulate Mitochondrial Structure-Function in Health and Disease.

Mitochondria are the energy-producing organelles within a cell. Furthermore, mitochondria have a role in maintaining cellular homeostasis and proper calcium concentrations, building critical components of hormones and other signaling molecules, and controlling apoptosis. Structurally, mitochondria are unique because they have 2 membranes that allow for compartmentalization. The composition and molecular organization of these membranes are crucial to the maintenance and function of mitochondria. In this review, we first present a general overview of mitochondrial membrane biochemistry and biophysics followed by the role of different dietary saturated and unsaturated fatty acids in modulating mitochondrial membrane structure-function. We focus extensively on long-chain n-3 (ω-3) polyunsaturated fatty acids and their underlying mechanisms of action. Finally, we discuss implications of understanding molecular mechanisms by which dietary n-3 fatty acids target mitochondrial structure-function in metabolic diseases such as obesity, cardiac-ischemia reperfusion injury, obesity, type 2 diabetes, nonalcoholic fatty liver disease, and select cancers.

Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome.

Cardiac mitochondrial phospholipid acyl chains regulate respiratory enzymatic activity. In several diseases, the rodent cardiac phospholipidome is extensively rearranged; however, whether specific acyl chains impair respiratory enzyme function is unknown. One unique remodeling event in the myocardium of obese and diabetic rodents is an increase in docosahexaenoic acid (DHA) levels. Here, we first confirmed that cardiac DHA levels are elevated in diabetic humans relative to controls. We then used dietary supplementation of a Western diet with DHA as a tool to promote cardiac acyl chain remodeling and to study its influence on respiratory enzyme function. DHA extensively remodeled the acyl chains of cardiolipin (CL), mono-lyso CL, phosphatidylcholine, and phosphatidylethanolamine. Moreover, DHA lowered enzyme activities of respiratory complexes I, IV, V, and I+III. Mechanistically, the reduction in enzymatic activities were not driven by a dramatic reduction in the abundance of supercomplexes. Instead, replacement of tetralinoleoyl-CL with tetradocosahexaenoyl-CL in biomimetic membranes prevented formation of phospholipid domains that regulate enzyme activity. Tetradocosahexaenoyl-CL inhibited domain organization due to favorable Gibbs free energy of phospholipid mixing. Furthermore, in vitro substitution of tetralinoleoyl-CL with tetradocosahexaenoyl-CL blocked complex-IV binding. Finally, reintroduction of linoleic acid, via fusion of phospholipid vesicles to mitochondria isolated from DHA-fed mice, rescued the major losses in the mitochondrial phospholipidome and complexes I, IV, and V activities. Altogether, our results show that replacing linoleic acid with DHA lowers select cardiac enzyme activities by potentially targeting domain organization and phospholipid-protein binding, which has implications for the ongoing debate about polyunsaturated fatty acids and cardiac health.

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars.

Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%-30% in case of women with stage III-IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model.

Dendritic cell activation, phagocytosis and CD69 expression on cognate T cells are suppressed by n-3 long-chain polyunsaturated fatty acids.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are bioactive n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in fish oil that exert immunosuppressive effects. A significant amount of literature shows that n-3 LCPUFAs suppress dendritic cell (DC) function in vitro; however, few studies have determined if the effects are emulated at the animal level. In this study, we first focused on the functional consequences of 5% (weight/weight) fish oil on splenic CD11c(+) DCs. Administration of n-3 LCPUFAs, modelling human pharmacological intake (2% of total kcal from EPA,1·3% from DHA), to C57BL/6 mice for 3 weeks reduced DC surface expression of CD80 by 14% and tumour necrosis factor-α secretion by 29% upon lipopolysaccharide stimulation relative to a control diet. The n-3 LCPUFAs also significantly decreased CD11c(+) surface expression and phagocytosis by 12% compared with the control diet. Antigen presentation studies revealed a 22% decrease in CD69 surface expression on transgenic CD4(+) T lymphocytes activated by DCs from mice fed fish oil. We then determined if the functional changes were mechanistically associated with changes in lipid microdomain clustering or plasma membrane microviscosity with n-3 LCPUFAs, as reported for B and T lymphocytes. Fish oil administration to mice did not influence cholera-toxin induced lipid microdomain clustering or microviscosity, even though EPA and DHA levels were significantly elevated relative to the control diet. Overall, our data show that n-3 LCPUFAs exert immunosuppressive effects on DCs, validating in vitro studies. The results also show that DC microdomain clustering and microviscosity were not changed by the n-3 LCPUFA intervention used in this study.

Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease.

Intracellular chloride channel protein CLIC1 regulates macrophage function through modulation of phagosomal acidification.

Intracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox- and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1(-/-) mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1(-/-) macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC1(-/-) mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs.

Mitochondrial permeability transition in the diabetic heart: contributions of thiol redox state and mitochondrial calcium to augmented reperfusion injury.

Mitochondria from diabetic hearts are sensitized to mitochondrial permeability transition pore (PTP) opening, which may be responsible for the increased propensity for cardiac injury in diabetic hearts. The purpose of this study was to determine if redox-dependent PTP opening contributes to augmented injury in diabetic hearts, and if compounds targeted at mitochondrial PTP, ROS, and calcium influx protected diabetic hearts from injury. Hearts from control or streptozotocin-induced diabetic rats were excised for either whole-heart or isolated mitochondria experiments. Myocardial glutathione content was oxidized in diabetic hearts when compared to control, and this translated to increased oxidation of the adenine nucleotide translocase in diabetic hearts. Diabetic mitochondria displayed significantly greater sensitivity to PTP opening than non-diabetic counterparts, which was reversed with the thiol-reducing agent dithiothreitol. The thiol-oxidant diamide increased calcium sensitivity in control, but not diabetic mitochondria. Diabetic animals treated with the mitochondria-targeted ROS suppressing peptide MTP-131 also showed improved resistance to PTP opening. In separate experiments hearts underwent ex vivo ischemia/reperfusion (IR). Diabetic hearts were more susceptible to IR injury, with infarct sizes of 60 ± 4% of the area-at-risk (vs. 46 ± 2% in non-diabetics; P<0.05). Administration of the PTP blocker NIM811 (5 µM), MTP-131 (1 nM) or the mitochondrial calcium uniporter blocker minocycline (1 µM) at the onset of reperfusion reduced infarct sizes in both control and diabetic hearts. These findings suggest that augmented susceptibility to injury in the diabetic heart is mediated by redox-dependent shifts in PTP opening, and that three novel mitochondria-targeted agents administered at reperfusion may be suitable adjuvant reperfusion therapies to attenuate injury in diabetic patients.

High doses of ketamine-xylazine anesthesia reduce cardiac ischemia-reperfusion injury in guinea pigs.

Choosing an appropriate anesthetic protocol that will have minimal effect on experimental design can be difficult. Guinea pigs have highly variable responses to a variety of injectable anesthetics, including ketamine-xylazine (KX). Because of this variability, supplemental doses often are required to obtain an adequate plane of anesthesia. Our group studies the isolated guinea pig heart, and we must anesthetize guinea pigs prior to harvesting this organ. In this study, we sought to determine whether a higher dose of KX protected isolated guinea pig hearts against myocardial ischemia-reperfusion injury. Male Hartley guinea pigs (Crl:HA; 275 to 300 g; n = 14) were anesthetized with either of 2 doses of KX (K: 85 mg/kg, X: 15 mg/kg; or K: 200 mg/kg, X: 60 mg/kg). After thoracotomy, hearts underwent 20 min of ischemia followed by 2 h of reperfusion. The high dose of KX significantly reduced myocardial infarct size as compared with the low dose (36% ± 3% and 51% ± 6%, respectively). Furthermore, the high dose of KX improved hemodynamic function over that associated with the low dose as measured by increases in both left ventricular developed pressure (49 ± 4 and 30 ± 8 mm Hg, respectively) and maximal rate of left ventricular relaxation (-876 ± 70 and -576 ± 120 mm Hg/s, respectively). However, the high dose of KX did not alter the maximal rate of left ventricular contraction or coronary flow. These results suggest that supplementation of KX to ensure an adequate anesthetic plane may introduce unwanted variability in ischemia-reperfusion studies.

PIP(2)-dependent inhibition of M-type (Kv7.2/7.3) potassium channels: direct on-line assessment of PIP(2) depletion by Gq-coupled receptors in single living neurons.

The open state of M(Kv7.2/7.3) potassium channels is maintained by membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). They can be closed on stimulating receptors that induce PI(4,5)P(2) hydrolysis. In sympathetic neurons, closure induced by stimulating M1-muscarinic acetylcholine receptors (mAChRs) has been attributed to depletion of PI(4,5)P(2), whereas closure by bradykinin B(2)-receptors (B2-BKRs) appears to result from formation of IP(3) and release of Ca(2+), implying that BKR stimulation does not deplete PI(4,5)P(2). We have used a fluorescently tagged PI(4,5)P(2)-binding construct, the C-domain of the protein tubby, mutated to increase sensitivity to PI(4,5)P(2) changes (tubby-R332H-cYFP), to provide an on-line read-out of PI(4,5)P(2) changes in single living sympathetic neurons after receptor stimulation. We find that the mAChR agonist, oxotremorine-M (oxo-M), produces a near-complete translocation of tubby-R332H-cYFP into the cytoplasm, whereas bradykinin (BK) produced about one third as much translocation. However, translocation by BK was increased to equal that produced by oxo-M when synthesis of PI(4,5)P(2) was inhibited by wortmannin. Further, wortmannin 'rescued' M-current inhibition by BK after Ca(2+)-dependent inhibition was reduced by thapsigargin. These results provide the first direct support for the view that BK accelerates PI(4,5)P(2) synthesis in these neurons, and show that the mechanism of BKR-induced inhibition can be switched from Ca(2+) dependent to PI(4,5)P(2) dependent when PI(4,5)P(2) synthesis is inhibited.

Anti-inflammatory activity in skin by biomimetic of Evodia rutaecarpa extract from traditional Chinese medicine.

BACKGROUND: Wu-Zhu-Yu, is an extract prepared from the small berry fruit of Evodia rutaecarpa and is reported to have anti-inflammatory and anti-nociceptic activity. Methyl nicotinate (MN) is known to induce the release of PGD(2) resulting in localized erythema within 30 min after topical application to human skin. OBJECTIVE: The purpose of this study was to determine if a defined biomimetic mixture of components of Evodia fruit extract inhibit inflammation in human cells and skin. METHODS: In order to control the potency of the test article, we prepared a defined biomimetic mixture of synthetic and natural forms of the active components of Evodia fruit extract, containing rutaecarpine, dehydroevodiamine, and evodin. This was tested for anti-inflammatory activity in UVB-irradiated cultured cells and in the MN model of micro-inflammation in human skin. RESULTS: This Evodia biomimetic mixture was a potent inhibitor of UVB-induced PGE(2) released by keratinocytes in culture. We found that MN also induces release of nitric oxide from cultured keratinocytes and microvascular endothelial cells. Twice daily application of 0.1-1% Evodia biomimetic mixture for 2 weeks significantly inhibited erythema after a MN challenge. A single application of 1% Evodia biomimetic mixture also significantly inhibited MN-induced erythema when applied at 60 min before, or within 5 min after MN exposure. The Evodia biomimetic mixture was significantly more effective at inhibiting erythema than bisabolol, the active component of chamomile. CONCLUSIONS: These results demonstrate that compounds found in E. rutaecarpa (including the indole quinazoline alkaloids) have powerful anti-inflammatory activity when applied topically to human skin.

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.